Carbon 13 nuclear magnetic resonance studies of Lactobacillus casei thymidylate synthetase containing biosynthetically incorporated [guanidino-13C]arginine.
Thymidylate synthetase, containing [guanidino-13C]arginine, was obtained from amethopterin-resistant Lactobacillus casei grown on a defined medium containing [guanidino-13C]arginine. Carbon 13 nuclear magnetic resonance spectroscopy was used to investigate the native enzyme and the response of 13C-enriched arginyl residues to binary and ternary complex formation and to chemical modification by 2,3-butanedione. Native enzyme exhibited four resonances (A, 158.3 ppm; B, 157.4 ppm; C, 156.9 ppm; and D, 156.1 ppm) in a chemical shift range of 2.2 ppm. The 12 arginyl residues per subunit of this enzyme were found in a 1:5:5:1 distribution in the four resonances. Carbon 13 NMR spectra revealed that of the four resonances, resonance C was most perturbed by binary and ternary complex formation. The ternary complex formed with [guanidino-13C]arginine-enriched enzyme, 5-fluoro-deoxyuridylate, and 5,10-methylenetetrahydrofolate exhibited two new resonances at 157.9 and 156.5 ppm. Carbon 13 NMR analysis of enzyme inactivated with butanedione yielded three new single-carbon resonances, one from resonance B and two from resonance C, demonstrating that three arginyl residues per subunit were derivatized. The results of the binary and ternary complex studies and the chemical modification experiment suggest that the active site arginyl residue is represented in resonance C of the native enzyme.
D001120:Arginine; D002247:Carbon Isotopes; D003931:Diacetyl / Q000494:pharmacology; D007780:Lacticaseibacillus casei / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D008780:Methyltransferases / Q000378:metabolism; D011487:Protein Conformation; D013940:Thymidylate Synthase / Q000378:metabolism
D002247:Carbon Isotopes; D001120:Arginine; D008780:Methyltransferases; D013940:Thymidylate Synthase; D003931:Diacetyl